TREATMENT OF lge-MEDIATED DISEASE

ABSTRACT

The methods and compositions described herein are based, in part, on the discovery of a polypeptide of soluble CD23 (sCD23) that binds and sequesters IgE. Thus, the sCD23 peptides, polypeptides and derivatives described herein are useful for treating conditions or disorders involving increased IgE levels such as e.g., allergy, anaphylaxis, inflammation, lymphoma, and certain cancers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. Ser. No.13/512,949, filed on Oct. 29, 2012, which is a a 35 U.S.C. §371 NationalPhase Entry Application of International Application No.PCT/US2010/058531, filed Dec. 1, 2010, which designates the UnitedStates, and which claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/265,439, filed Dec. 1, 2009 the contentsof which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 11, 2012, isnamed 066742US.txt and is 149,050 bytes in size.

GOVERNMENT SUPPORT

This invention was made with Government support under Contract No.AI074843 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The field of the invention relates to the treatment of IgE mediateddisease.

BACKGROUND

Allergy is a chronic inflammatory disease that encompasses a broad rangeof manifestations including allergic rhinitis (AR), eczema, and asthma.It has been approximated that up to 25% (75 million) of the USpopulation suffers from some form of allergy. In fact, seasonal AR isestimated to affect 25% of the Caucasian race of which 40% are children.The prevalence of asthma is estimated to be 9% (27 million people) inthe USA. Food allergies are on the rise and are particularly dangerousin young children as they have a propensity to develop into lifethreatening conditions, such as anaphylactic shock.

60 million Americans suffer from AR in the US. AR is characterized bynasal congestion, rhinorrhea (runny nose), sneezing, and nasal and eyeitchiness. AR can be classified as a nuisance condition but it isassociated with significant morbidity that often results in a reducedquality of life, emotional well-being and productivity. Most of thereduction in the quality of life can be attributed to sleepdisturbances. The burden is heavy on children as 88% of pediatricpatients with AR have difficulty sleeping.

In fact, AR in particular, places a considerable economic burden on theUS health care system which includes direct costs to both patients andinsurance providers and indirect costs such as absenteeism andpresenteeism. Further, children with AR are more likely to haveincreased visits to their physicians further increasing the cost ofhealth care.

SUMMARY OF THE INVENTION

The methods and compositions described herein are based, in part, on thediscovery of a polypeptide of soluble CD23 (sCD23) that binds andsequesters IgE to induce an anti-inflammatory response. Thus, the sCD23polypeptides, peptides and derivatives described herein are useful fortreating conditions or disorders involving increased IgE levels such ase.g., allergy, anaphylaxis, inflammation, lymphoma, and certain cancers.

In one aspect, provided herein are compounds of the formula X₁—R,wherein R comprises SEQ ID No. 4 and X₁ comprises at least threecontiguous amino acids of the sequence

TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 7), andcontiguous fragments thereof. In one embodiment, X₁ consists of thesequence TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:7). In one embodiment, X₁ comprises at least 5 contiguous nucleotides ofthe sequence TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:7). In other embodiments, X₁ comprises or consists of at least 7, atleast 9, at least 10, at least 12, at least 15, at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 55, at least 60, at least 65, at least 70, at least 75, at least80, at least 85, at least 90, at least 95, at least 100, at least 105contiguous amino acids of the sequence TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 7). In one embodiment, thefragment ends with the carboxy sequence LQVS (SEQ ID NO: 8). In oneembodiment conservative amino acid substitutions as set forth herein canbe made.

In one embodiment of this aspect, the compound binds IgE. In anotherembodiment of this aspect, the compound does not bind CD21.

In another embodiment of this aspect, the compound comprises a highaffinity binding site for IgE. In another embodiment, the high affinitybinding site is generated by amino acid substitution of R. In anotherembodiment, the amino acid substitution is D107E.

Also provided herein in another aspect are compounds of the formulaR—X₂, wherein R comprises SEQ ID No. 4 and X₂ comprises SEGSAE (SEQ IDNO: 9), SEGSA (SEQ ID NO: 10), SEGS (SEQ ID NO: 11), SEG, SE, S, L,—COOH. In one embodiment, the compound is R₀. In one embodiment, thecompound can have the formula X₁—R₀—X₂, wherein X₁ and X₂ are as definedherein. In one embodiment of this aspect, the compound binds IgE. Inanother embodiment of this aspect, the compound does not bind CD21.

In another embodiment of this aspect, the compound comprises a highaffinity binding site for IgE. In another embodiment, the high affinitybinding site is generated by amino acid substitution of R. In anotherembodiment, the amino acid substitution is D107E (e.g., SEQ ID No: 2).

In one embodiment the compound is PEGylated.

Another aspect described herein relates to compositions for sequesteringIgE, the composition comprising: an effective amount of a compound asdescribed above and a pharmaceutically acceptable carrier.

Also provided herein are methods for reducing a subject's immuneresponse to an allergen, the method comprising: administering to asubject a pharmaceutical composition containing an effective amount of acompound of formula X₁—R₀—X₂, R₀—X₂, or X₁—R₀, and a pharmaceuticallyacceptable carrier.

In one embodiment, the pharmaceutical composition is administeredprophylactically to a subject at risk of having an immune response to anallergen.

In another embodiment of this aspect, the pharmaceutical composition isadministered to a subject following exposure to the allergen.

In another embodiment of this aspect, the method further comprisesadministering steroid therapy.

In another embodiment of this aspect, the method further comprisesadministering allergy shots to said individual.

In another embodiment of this aspect, the allergen is a food allergen, apollen, a plant allergen, a dust mite, animal dander, insect stings, afungus, a spore, a mold, latex, or a drug.

In another embodiment of this aspect, the method further comprises astep of selecting an individual having an immune response to anallergen.

Also provided herein are methods for treating an IgE-mediated disease ina subject, the method comprising administering to a subject apharmaceutical composition containing an effective amount of a compoundof formula X₁—R₀—X₂, R₀—X₂, or X₁—R₀, and a pharmaceutically acceptablecarrier.

In one embodiment of this aspect, the IgE mediated disease is selectedfrom the group consisting of: allergy, anaphylaxis, asthma, eczema, andrhinitis

Also provided herein are methods for reducing the development of achronic immune response to an allergen, the method comprisingadministering to a subject a pharmaceutical composition containing aneffective amount of a compound of formula X₁—R₀—X₂, R₀—X₂, or X₁—R₀, anda pharmaceutically acceptable carrier.

Also provided herein are methods for reducing the dose of an allergy oranaphylaxis treatment, the method comprising administering to a subjectbeing treated with an allergy or anaphylaxis treatment a pharmaceuticalcomposition containing an effective amount of a compound of formulaX₁—R₀—X₂, R₀—X₂, or X₁—R₀, and a pharmaceutically acceptable carrier.

Definitions

A “subject” in the context of the present invention is preferably amammal. The mammal can be a human, non-human primate, mouse, rat, dog,cat, horse, or cow, but is not limited to these examples.

As used herein, the term “sCD23 peptide” or “sCD23 polypeptide” refersto a modified peptide or polypeptide of soluble CD23 comprising, at aminimum, amino acid residues 156-292 of sCD23. The term “sCD23 peptide”or “sCD23 polypeptide” also encompasses peptides or polypeptides havingconservative substitution mutations, mutations to enhance IgE bindingactivity, and/or mutations to enhance calcium binding activity. Incertain embodiments, the term sCD23 peptide or polypeptide refers to apolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a derivative thereof.The terms peptide and polypeptide are used herein to denote proteinfragments of different lengths, wherein the term “peptide” refers toshort amino acid sequences and polypeptide refers to longer amino acidsequences. The use of sCD23 peptides and polypeptides are bothcontemplated for use with the methods and compositions described herein.One of skill in the art will understand the terms “peptide” and“polypeptide.” One need not know the exact length of an sCD23 peptide orpolypeptide since both are contemplated for use with the methodsdescribed herein.

The term “derivative” as used herein refers to peptides or polypeptideswhich have been chemically modified, for example by ubiquitination,labeling, pegylation (derivatization with polyethylene glycol) oraddition of other molecules. A molecule is also a “derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties can improve themolecule's solubility, absorption, biological half life, etc. Themoieties can alternatively decrease the toxicity of the molecule, oreliminate or attenuate an undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl.,Easton, Pa. (1990). A derivative of an sCD23 peptide/polypeptide willsubstantially retain the IgE binding activity of thepeptide/polypeptide, that is, the derivative will retain at least 20% ofthe IgE binding activity of the parent peptide/polypeptide as measuredusing e.g., an in vitro immunoprecipitation activity assay as describedherein in the Example section. In one embodiment, the peptide orpolypeptide is PEGylated.

As used herein, the term “PEGylated” refers to a polyethylene glycolderivative attached to a peptide or polypeptide as described herein.

As used herein, the term “substantially retains IgE binding activity”means that a derivative will retain at least 30% of the IgE bindingactivity (as assessed by an in vitro immunoprecipitation assay asprovided herein in the Examples section) of the polypeptide or peptidefrom which it is derived. In other embodiments, the derivative willretain 3 at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99% or even 100% of theIgE binding activity of the peptide/polypeptide from which it isderived. The term “substantially retains IgE binding activity” alsoencompasses an increase in the IgE binding activity of a derivativecompared to that of the parent peptide/polypeptide, for example, thederivative can have at least a 2-fold increase, at least 5-fold, atleast 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, atleast 1000-fold or more increase in IgE binding activity compared to theparent peptide/polypeptide from which it is derived. IgE binding assaysfor assessing IgE binding activity are well known in the art (see e.g.,Chen et al. J Immunological Methods 58(1-2):59-71 (1983); Matsuo et al.J Immunology 175:8116-8122 (2005)).

As used herein, the term “lacks substantial CD21 binding” refers to thelack of detectable binding of an sCD23 polypeptide to CD21 using e.g.,an in vitro binding assay. As used herein, the term “reduced CD21binding” refers to a decrease in the level of CD21 binding to a modifiedsCD23 polypeptide of at least 10% (as measured using e.g., an in vitroCD21 binding assay) compared to native sCD23; preferably the level ofCD21 binding is reduced at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99% or even 100% (i.e., below detectable levelsusing a standard CD21 binding assay).

As used herein, the term “immune response to an allergen” is used todescribe an increase in IgE levels in a subject and/or a subject'sresponse to increased IgE levels, and can present with such symptoms assneezing, coughing, sinus congestion, mucus production in the sinuses(rhinitis) or lungs (asthma).

As used herein, the term “increase in IgE levels” refers to an increasein the level of IgE in a subject of at least 10% following exposure toan allergen compared to the level of IgE prior to allergen exposure. Inother embodiments, an “increase in IgE levels” refers to an increase inIgE in a subject exposed to an allergen of at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 1-fold, at least 5-fold, at least10-fold, at least 100-fold, at least 500-fold, at least 1000-fold, atleast 5000-fold, at least 10,000-fold or more compared to the level ofIgE in the subject prior to allergen exposure. Allergen exposure can bespontaneous, in that the subject comes into contact with an allergen,for example, in a particular environment, by ingestion, or during aparticular season. Alternatively, an allergen can be actively deliveredin a clinical setting such as e.g., skin tests for allergy.

As used herein, the term “administered prophylactically” refers toadministration of a peptide or polypeptide to a subject prior exposureto an allergen expected to promote an increase in IgE levels, forexample prior to allergen exposure. Administration can occur e.g.,minutes before an expected exposure, or on e.g., a daily, weekly,bi-weekly, or monthly basis. An appropriate treatment regime can bedetermined by one of skill in the art. Prophylactic administration alsoencompasses co-administration with other agents such as e.g., allergyshots, or steroid injections.

As used herein, the term “following exposure to the allergen” refers toadministration following exposure to an allergen including withinseconds, minutes, or hours following a subject's exposure to anallergen, or when at least one symptom of IgE mediated disease ispresent in a subject. Thus, the methods and compositions used herein areuseful in the treatment of both an acute exposure to an allergen andchronic (e.g., seasonal) exposure to an allergen.

As used herein, the term “selecting an individual having an immuneresponse to an allergen” refers to a step of first diagnosing a subjectas having an IgE mediated response to an allergen prior toadministration of a modified sCD23 peptide or polypeptide. Diagnosis ofan allergic response can be performed using any number of techniquesknown to those of skill in the art, such as e.g., skin prick test,intradermal test, skin patch test, blood test (e.g., ELISA, immunoassaycapture test, radioallergosorbent test), among others.

As used herein, the term “IgE mediated disease” refers to a disease thatis mediated, at least in part, by an increase in the levels of IgE asthat term is used herein.

As used herein, the term “chronic immune response to an allergen” refersto an IgE mediated response that develops into a chronic condition ordisease, such as asthma, or eczema. The term “chronic immune response”is not intended to encompass a single, acute exposure to an allergene.g., insect stings, poison oak, etc.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows depiction of cell bound CD23 cleavage to produce variouspolypeptides of soluble CD23.

FIGS. 2A-2C show characterization of sCD23 in supernatants of B cellstreated with schistosome antigens. FIG. 2A shows that an ˜15 kDa bandaccumulates in B cell culture supernatants in the presence ofschistosomal egg protein (SEA). Human sera contains multiple sizes ofCD23 but generally lack the 15 kDa protein. FIG. 2B shows the increasein the 15 kDa band by SEA or SWAP was decreased by the presence of sCD21indicating that sCD21 blocks a cleavage site. FIG. 2C shows thatSWAP-treatment enhances the binding of free IgE to low MW sCD23. SWAP:soluble worm antigen preparation from schistosomes.

FIG. 3 shows generation of a 15 kDa sCD23 protein in a parasitic diseasein which the protein was discovered. Schistosomes modify sCD23 in amanner that retains the IgE binding but reduces the CD21-bindingability.

FIG. 4 shows application of schistosome-modified sCD23 for binding IgE.

FIGS. 5A-5I show depiction of the advantage of modified sCD23 inreducing allergic responses. FIG. 5A shows sensitized mast cells at amucosal surface (lung or gut) poised to respond to allergens. In FIG. 5Bcircles demonstrate antigen-specific region of IgE antibody. FIG. 5C.Antigen-binding to the cell-bound IgE “cross-links” the IgE. FIG. 5Dshows that cross-linked IgE induces cellular degranulation which causesallergic symptoms. FIG. 5E shows that cortico-steroids inhibit theproduction of IgE and other inflammatory events. FIG. 5F shows thatanti-histamines, decongestants, and leukotriene inhibitors reduce theeffects of degranulation following IgE cross-linking. FIG. 5G showsmechanism of Xolair which binds the non-specific Fc region of IgE andprevents IgE from binding to its cellular receptors. FIG. 5H shows thatdue to its binding properties, Xolair has the potential to globallycross-link cell-bound IgE. Because Xolair binds IgE in the non-specificregion, it has the potential to globally induce a potent allergicresponse from the activated cells. FIG. 5I shows that mechanism ofaction of modified sCD23 which binds the Fc region of IgE (with loweraffinity) and inhibits IgE binding to IgE receptors. Because of itsstructure, the modified sCD23 polypeptide has a very low potential tocross-link cell bound IgE non-specifically and thus presents a low riskof inducing anaphylaxis and other severe adverse effects.

FIG. 6 shows a schematic diagram relating to the production of modifiedsCD23. Image shows a schematic representation of the expression vectorused to produce sCD23 peptides. Selection and expression controlcomponents are highlighted. The sCD23 sequences were cloned into thepET-28a(+) vector and propagated in Top10F media. For expressionpurposes, BLR DE3 bacteria were transformed and selected on Kan plates.Colonies were chosen for protein production using standard protocols.

FIGS. 7A and 7B show data indicating that the expressed protein is atthe correct predicted molecular weight. FIG. 7A shows modified sCD23 wasexpressed as described in FIG. 6. Shown is a protein fragmentrepresented in SEQ ID No. 1, containing a c-myc peptide tag and a 6-his(SEQ ID NO: 12) affinity purification tag. Following expression, proteinwas recovered using nickel chromatography. Proteins were then separatedby SDS-PAGE and the resulting purified protein is shown in Lane 3. FIG.7B shows Western blot analysis using an anti-myc antibody identifies aband of the same molecular weight as the coomassie-stained band in 7A,lane 3. Notable is that all fractions contain the protein as expected.Thus, the sequence used to generate the modified sCD23 protein generatesa highly similar protein to the fragment generated by schistosomes.Further, this demonstrates that the sCD23 protein fragments are easilyexpressed thereby facilitating the required increase in scalability forclinical trials and patient treatment.

FIG. 8 is a schematic showing a high affinity modified sCD23, anexemplary modification of the sCD23 protein.

FIG. 9 is a micrograph indicating that modified sCD23 is found in serumof patients with schistosomiasis. The 15 kDa sCD23 fragment is apparentin patients hyper-exposed to infectious schistosomes in Western Kenya(top panel). In contrast, subjects unexposed/uninfected rarelydemonstrate the fragment (lower panel).

FIGS. 10A and 10B are graphs showing in vitro data demonstratingmechanistically that schistosome modified sCD23 acts as a decoy receptorfor IgE and prevents IgE from binding to its cell surface receptorFcεRIα. FIG. 10A shows recombinant full length CD23 exposed toschistosome antigens inhibits exogenous IgE from binding toIL-10-treated THP-1 cells (left panel). IL-10 increases FcαRIα chainexpression, the high affinity IgE receptor, by THP-1 cells. No IgE: grayfill; 1 μg/ml IgE; IgE+modified sCD23 (1 μg/ml). THP-1 cells wereexposed to IgE and sCD23 and rotated at 4° C. for 2 hours. Surface IgEwas measured by flow cytometry. Modified sCD23 antigen prep containedboth full length and 15 kDa protein fragments. FIG. 10B shows thatunmodified rsCD23 does not inhibit IgE binding compared to IgE alone. NoIgE shown as a gray fill.

DETAILED DESCRIPTION

Described herein are methods and compositions useful for treating IgEmediated conditions or diseases comprising administering to a subject inneed thereof, a modified peptide (or polypeptide) of soluble CD23 thatbinds IgE and in some embodiments lacks substantial CD21 binding. Theinvention is based, in part, on the discovery that soluble polypeptidesof CD23 can bind and sequester IgE, prevent IgE binding to its receptor,thereby reducing an immune response initiated by IgE. The methods andcompositions described herein are useful for treating IgE mediatedconditions or diseases such as e.g., allergic diseases, lymphomas,inflammatory diseases and certain cancers.

IgE Mediated Disease

The methods and compositions described herein are useful for treatingany IgE mediated disease, that is a condition or disorder characterizedby increased levels of IgE. Such disorders include, for example,allergic diseases such as rhinitis, eczema, food allergy, asthma, andanaphylactic shock; lymphoma; pancreatic cancer; cystic fibrosis; celiacdisease; and chronic uticaria.

One of skill in the art can easily determine if a subject has an IgEmediated disease by measuring the IgE levels in a biological sample(e.g., blood sample) from the subject or by assessing such symptoms assneezing, coughing, mucus production, skin irritation, redness,swelling, difficulty breathing, itchiness, among others followingexposure to an allergen.

In one embodiment, the IgE mediated disease comprises an allergicdisease (e.g., an immune response to an allergen). Some common allergensthat produce allergic disease in subjects include e.g., food allergens(e.g., nuts, soy, dairy, gluten, eggs, seafood), pollen (e.g., fromgrass, trees, weeds), plant product allergens (e.g., poison oak, poisonivy), dust mite excretions (e.g., feces, chitin), animal dander (e.g.,cat, dog, rabbit hair), insect stings (e.g., bee, wasp, ant, mosquito),fungus, spores (e.g., mushroom spore), mold, latex, metal, or drugs(e.g., penicillin, sulfonamides).

The Role of IgE in Mediating Allergic Responses

Allergy results when a subject's immune system reacts inappropriately toharmless molecules, such as pollen, dust mite and nuts. In general,allergy is mediated primarily by certain components of the immune systemincluding IgE, mast cells, basophils and eosinophils. IgE is produced byB cells, circulates in the blood and binds to IgE-specific receptors(FcεRI) on the surface of mast cells and basophils. Allergens can thenbind to the IgE on the IgE-coated cells. Cross-linking of the IgE and Fcreceptors activates the cells, which causes degranulation. Degranulationreleases histamine and other inflammatory chemical mediators such asleukocytes and prostaglandins. These mediators cause several systemiceffects, such as vasodilation, copious mucus production, and smoothmuscle contraction. This results in the runny nose, itchiness, dyspnea,and potentially anaphylaxis associated with allergy. Depending on theindividual, allergen, and mode of introduction, the symptoms can besystem-wide (classical anaphylaxis), or localized to particular bodysystems. For example, asthma is localized to the respiratory system andeczema is localized to the skin.

Current Treatment and Management of Chronic Allergic Disease

The continued high burden of allergic disease and increased prevalenceof food allergy and asthma indicates suboptimal control despite avariety of pharmacological and non-pharmacological treatments. Ingeneral, patients report low satisfaction with allergy treatments.Intranasal corticoid steroids (INCS) are considered the gold-standardfor AR but this treatment is associated with bone mineral lost, growthretardation, and adrenal suppression which is troublesome for children.Over the counter (OTC) anti-histamine/decongestants also have adverseeffects, such as drowsiness, epistaxis, and burning and have thetendency to stop working. The burden on health care and morbidityassociated with allergy warrants the development of new strategies toalleviate chronic allergy.

Current Anti-IgE Therapy

Omalizumab (Xolair) is a humanized monoclonal antibody specific for theregion of IgE that binds the high affinity IgE receptor on effectorcells (mast cells and basophils). Subcutaneous administration reducesthe ability to detect serum IgE within hours and reduces the number ofhigh affinity receptors over 8-12 weeks. Several allergic diseases havebeen found to respond well to Xolair. Asthma attacks can be reduced19-75% with Xolair added to corticosteroid therapy. Further, in somestudies, patients treated with Xolair were able to reduce their dailycorticosteroid dosage or stop use altogether. Xolair is more expensivethan other current asthma treatments and health and reimbursementauthorities are increasingly demanding evidence of economic benefit tosupport pricing and formulary listing. Thus, given its high cost, therequirement that it be administered in a physician's office, and sideeffects, it is appropriate for only a small percentage of patients withasthma. Further, because of the nature of the molecular interaction ofthis treatment, it can cause anaphylactic shock, a dangerous allergicreaction, in some patients. The methods and compositions describedherein offer an effective, low-cost, safe alternative with little sideeffects, based on a similar mechanism to that of Xolair.

Role of CD23 in Immunity

CD23 has a broad cellular distribution and in addition to B cells, isexpressed on monocytes, resting eosinophils, and follicular dendriticcells. CD23 also has multiple ligands other than IgE, including CD21 andMac-1. CD23 is a type II integral membrane protein with acalcium-dependent lectin domain in the C-terminal end of theextracellular region that binds IgE. A leucine zipper in the N-terminalregion allows the CD23 molecules at the cell surface to formhomo-trimers which increase the affinity for IgE to the same level asthe FcεRI. The C-terminal tail, which “hangs off” the globular lectinhead has been shown to bind CD21. There are two isoforms of human CD23:CD23a and CD23b. Without wishing to be bound by theory, both isoformsare thought to be regulated in part by IL-4, but act on separatetranscription initiation sites. CD23a and CD23b vary at the N-terminalcytoplasmic region only by a few residues, but possess identicalC-terminal extracellular regions. CD23a is expressed primarily by Bcells whereas CD23b is inducible by IL-4 and other stimuli on B cells,eosinophils, and monocytes.

Soluble (s)CD23: sCD23 exists in multiple forms and has numerous effectson the immune system. sCD23 results when surface CD23 (a or b) isproteolytically cleaved thereby releasing the extracellular region intothe microenvironment. Endogenous proteases cleave the cell-surfaceprotein near the base of the stalk to release a 37 kDa molecule and atsites closer to the head resulting in several different sized sCD23fragments. Human serum contains five different molecular weightfragments of monomeric CD23: 37, 33, 29, 25, and 16/17 kDa. Themembrane-bound metalloproteinase, ADAM10, catalyzes the cleavage offragments derived from two distinct sites in the CD23 backbone releasingthe 37 and 33 kDa peptides. Neutrophils secrete human leukocyte elastaseand cathepsin G, both which efficiently cleave CD23 on B cells. Theneutrophil elastase was shown to cleave the 37 kDa fragment into the 25kDa fragment though the fragment size(s) generated with cathepsin G wasnot described. The function of sCD23 on subsequent IgE synthesis andcellular activation depends upon whether sCD23 is an oligomer, large orsmall fragment, and to which ligands it binds (CD23-bound IgE, BCRε,CD21).

Differentially cleaved fragments of sCD23 influence immunity: Cleavageof cell surface CD23 in the N-terminal “stalk” by ADAM10 and otherproteases generates 29, 33 and 37 kDa fragments that retain the abilityto homo-trimerize (or retain the cell surface homotrimer structure). Thesmaller fragments, 16/17 and 25 kDa, are thought to be generated fromthe cleavage of the larger molecules by other host cysteine proteases.The level of sCD23 is regulated in part by IL-4 and IgE. Thus, treatmentof B cells with IL-4 increases the concentration of sCD23 insupernatants whereas the addition of IgE inhibits the release of sCD23.Even in its soluble form, CD23 has multiple ligands other than IgE,including CD21 (the complement 2 receptor) and Mac-1 and has pleitropicfunctions. sCD23-containing supernatants derived from B cell culturesinduce IgE production in the absence of T cells and IL-4.

Furthermore, sCD23 may rescue B cells from apoptosis by interacting withCD21 and perhaps lower the threshold of B cell receptor-mediatedactivation through cross-linking CD21. The 25 kDa fragment of sCD23 hasbeen shown to promote differentiation of germinal center B cells bybinding to CD21.

sCD23 trimeric clusters have high affinity for BCRε as well andstimulate IgE expression. In a more recent study, three recombinantfragments of CD23 were compared for their ability to stimulate IgEsynthesis from anti-CD40/IL-4 activated tonsillar B cells. Monomeric“derCD23” which contains the lectin head and a portion of the C-terminaltail (similar to that released by Derp 1) inhibited IgE synthesis. Incontrast, the “lz CD23”, which retained the leucine zipper to allow fortrimerization, stimulated IgE synthesis. The monomeric “exCD23”, whichlacked the leucine zipper but retained the full c-terminal tail, couldnot trimerize, and did not affect IgE production. The authorshypothesized that the lzCD23 was able to induce large networks ofcross-linked BCRε and CD21 on the cell surface (FIG. 1) therebyactivating the cell.

sCD23 has effects on other cells as well. The 25 kDa protein appears tobe pro-inflammatory and induces the secretion of IL-6, IL-1β and TNF-αfrom monocytes. Soluble fragments released by serine proteases ofneutrophils also stimulated resting monocytes to produce oxidative burstand proinflammatory cytokine without any co-stimulatory signal. Severalpatents relate to the inhibition of production of sCD23 and TNF-α (seee.g., U.S. Pat. Nos. 6,673,965 and 6,235,753).

Further complicating the immunobiology of CD23, CD21 is also found in asoluble form and binds both surface CD23 and sCD23. Soluble CD21 isspontaneously released by B cells upon shedding of the extracellulardomain of the molecule. CD23 recognizes two main epitopes on the CD21molecule. One region consists of short consensus repeat sequences (SCRs)1-2 and the other of SCRs 5-8 although the effect of CD23 binding to oneor either sites on the immune response have not been defined.

Microbial cleavage of surface CD23: Microbial proteases, such as dustmite allergen, Derp1, also cleave CD23. Derp1 is a cysteine proteasethat cleaves CD23 at two sites (Ser155-Ser156 and Glu298-Ser299) toproduce a 16/17-kDa fragment containing the lectin domain and part ofthe C-terminal tail (amino acids 156-298). This 16/17-kDa fragmentcontains the minimum structural requirement for binding to both IgE andCD21 but lacks the leucine zipper portion and thus does not oligomerize.

Schistosomes appear to cleave CD23 similarly with the major differenceapparent in the CD21 binding properties of the modified molecule.Whereas schistosome generated sCD23 appears to bind IgE, it lacks theability to bind CD21. The resulting polypeptide is approximately 15 kD.This schistosome-modified CD23 binds to free IgE and likely sequestersit from binding to high affinity receptors on effector cells, such asmast cells. The methods and compositions described herein are based, inpart, on these findings. Modified sCD23 represents a viable therapeuticto sequester IgE in a more natural and safe manner to prevent itsbinding to FcεRI and thus allergic responses.

sCD23 Polypeptide and Peptide Compounds

Peptides or polypeptides useful with the methods described hereincomprise modified peptides (or polypeptides) of a soluble form of CD23,and particularly sCD23 polypeptides or peptides that comprise a lectinIgE binding domain but lack a CD21 complement-binding domain. IgE bindsto the lectin-head region (including residues from (aa187 to 279), whileCD21 appears to involve residues that are separated in both primarystructure and tertiary structure (residues 294 and above). Thus, afragment from 156-292 includes the lectin binding region that isimportant for binding to IgE but is devoid of the residues required forCD21 interaction.

The CD21-binding tail encompasses amino acids 290 to 321. In oneembodiment, a modified sCD23 is a non-CD21 binding protein that lacksamino acids 293-321. CD21 complement binding requires at least aminoacids 290-298. Variations of this protein include producing sCD23lacking any amino acids from 290 to 321. The CD21 domain is a C-terminaldomain and at a minimum consists of amino acids 290-298 of SEQ ID NO: 3.In one embodiment, the sCD23 polypeptide comprises SEQ ID NO. 1, 2, 3,4, 5 or 6. In another embodiment, the sCD23 polypeptide consistsessentially of SEQ ID NO. 1, 2, 3, 4, 5 or 6. In another embodiment, thesCD23 polypeptide consists of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

SEQ ID No: 3 illustrated below depicts the lectin IgE binding domain theC-tail CD21 binding domain, and the start of the sCD23 polypeptide.

SEQ ID No: 3 MEEGQYSEIE ELPRRRCCRR GTQIVLLGLV TAALWAGLLT LLLLWHWDTTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ

ELQVSSGFVC N TCPEKWINF QRKCYYFGKG TKQWVHARYA CDDMEGQLVSIHSPEEQDFL TKHASHTGSW IGLRNLDLKG EFIWVDGSHV DYSNWAPGEP

GPDSRPDPDG RLPTPSAPLH S underlined text = lectin IgE binding domainitalicized text = C-tail CD21 binding domain bold text =start of common sCD23 polypeptide (172 aa)

Human soluble CD34 is modified by schistosomes (a protease cleavageevent), which produces a CD23 fragment that binds IgE, but not CD21.This novel CD23 fragment plays an important role in controllinginappropriate IgE mediated immunity in schistosomiasis and that it isthe IgE-binding characteristic only that is important. Thus, it iscontemplated herein in one embodiment that sCD23 peptides orpolypeptides useful with the methods and compositions described hereinwill bind IgE while lacking substantial CD21 binding.

It is important to note that preferred sCD23 peptides or polypeptideslack a CD21 binding site, since this site likely contributes to thepro-inflammatory activity of some sCD23 polypeptides. For example, ithas been previously reported by one group that sCD23 retains thepro-inflammatory properties of the full-length CD23 protein andincreases TNF-alpha production (see e.g., Daniels, B. et al., (2005)Cellular Immunity 234(2):146-153). Daniels et al. conclude that the“production of proinflammatory cytokines [by sCD23], particularly tumornecrosis factor-α will enhance immune responses in cases of asthma,allergy, and hyper-IgE syndrome.” However, Daniels et al. use arecombinant sCD23 150-321 fragment, which contains a CD21-bindingdomain. Binding of CD21, which is a complement receptor, is most likelythe mediator of cytokine production and other inflammatory effects.Additionally, this fragment may trimerize (due to its longer stalk),which can cross-link large networks of CD21 and CD23-bound IgE on thecell surface, thereby activating cells. Further, the sCD23 described byDaniels et al. binds IgE with low affinity. In some embodiments of themethods described herein, the preferred sCD23 fragment is 14-15 kDa andlacks the ability to trimerize and/or to bind CD21. In other embodimentsdescribed herein, a high affinity IgE binding sCD23 is preferred for usewith the methods described herein (i.e., a D258E mutation of sCD23; SEQID No.: 3). Such high affinity binding of sCD23 to IgE enhances thehalf-life of CD23-IgE complexes by at least 10% compared to non-modifiedsCD23 fragments. It is contemplated that the preferred sCD23 peptides orpolypeptides described herein do not cause inflammation (e.g., bybinding CD21 or producing trimers or multimers) and/or can compete forbinding sites of the more inflammatory fragments of sCD23, with anoverall result of reducing inflammation.

In some embodiments, the level of CD21 binding to an sCD23 polypeptideis reduced. In other embodiments, an sCD23 polypeptide substantiallylacks CD21 binding activity. CD21 binding activity can be reduced usingany method known to those of skill in the art. For example, the entireCD21 binding site (e.g., residues 294 and above) can be omitted duringsynthesis of the polypeptide or cleaved from the sCD23 polypeptide. Inother embodiments, one or more amino acid deletions, substitutions, oradditions can be utilized to disrupt the binding of CD21 to the sCD23polypeptide. One of skill in the art can easily test for a disruption ofCD21 binding using e.g., an in vitro binding assay or animmunoprecipitation-based assay.

In one aspect, the peptide compound has a formula of X₁—R₀, wherein R₀comprises SEQ ID No. 4 (or a derivative thereof such as e.g., SEQ ID No.4 having a D107E mutation) and X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS ISEQ ID NO: 7). In another embodiment ofthis aspect, X₁ comprises at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 22, at least24, at least 25, at least 27, at least 30, at least 35, at least 37, atleast 40, at least 42, at least 45, at least 47, at least 50, at least55, at least 60, at least 65, at least 70, at least 75, at least 80, atleast 85, at least 90, at least 95, at least 100, at least 105, or atleast 106 amino acids of SEQ ID NO. 4. For example, X₁ can compriseVTKLRM ELQVS (SEQ ID NO: 13). In another embodiment of this aspect, X₁comprises EVTKLRM ELQVS (SEQ ID NO: 14). In another embodiment of thisaspect, X₁ comprises EEVTKLRM ELQVS (SEQ ID NO: 15). In anotherembodiment of this aspect, X₁ comprises REEVTKLRM ELQVS (SEQ ID NO: 16).In another embodiment of this aspect, X₁ comprises LREEVTKLRM ELQVS (SEQID NO: 17). In another embodiment of this aspect, X₁ comprises RLREEVTKLRM ELQVS (SEQ ID NO: 18). In another embodiment of this aspect,X₁ comprises ER LREEVTKLRM ELQVS (SEQ ID NO: 19). In another embodimentof this aspect, X₁ comprises LER LREEVTKLRM ELQVS (SEQ ID NO: 20). Inanother embodiment of this aspect, X₁ comprises LLER LREEVTKLRM ELQVS(SEQ ID NO: 21). In another embodiment of this aspect, X₁ comprisesDLLER LREEVTKLRM ELQVS (SEQ ID NO: 22). In another embodiment of thisaspect, X₁ comprises SDLLER LREEVTKLRM ELQVS (SEQ ID NO: 23). In anotherembodiment of this aspect, X₁ comprises ASDLLER LREEVTKLRM ELQVS (SEQ IDNO: 24). In another embodiment of this aspect, X₁ comprises EASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 25). In another embodiment of this aspect,X₁ comprises NEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 26). In anotherembodiment of this aspect, X₁ comprises RNEASDLLER LREEVTKLRM ELQVS (SEQID NO: 27). In another embodiment of this aspect, X₁ comprises ERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 28). In another embodiment ofthis aspect, X₁ comprises NE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:29). In another embodiment of this aspect, X₁ comprises LNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 30). In another embodiment of this aspect,X₁ comprises ELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 31). Inanother embodiment of this aspect, X₁ comprises QELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 32). In another embodiment of this aspect,X₁ comprises SQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 33). Inanother embodiment of this aspect, X₁ comprises KSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 34). In another embodiment of this aspect,X₁ comprises FKSQELNE RNEASDLLER EEVTKLRM ELQVS (SEQ ID NO: 35). Inanother embodiment of this aspect, X₁ comprises SFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 36). In another embodiment of this aspect,X₁ comprises SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 37). Inanother embodiment of this aspect, X₁ comprises L SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 38). In another embodiment of this aspect,X₁ comprises DL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 39).In another embodiment of this aspect, X₁ comprises ADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 40). In another embodiment ofthis aspect, X₁ comprises QADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 41). In another embodiment of this aspect, X₁ comprisesLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 42). In anotherembodiment of this aspect, X₁ comprises GLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 43). In another embodiment of this aspect,X₁ comprises NGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:44). In another embodiment of this aspect, X₁ comprises LNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 45). In anotherembodiment of this aspect, X₁ comprises NLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 46). In another embodiment of this aspect,X₁ comprises WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ IDNO: 47). In another embodiment of this aspect, X₁ comprises S WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 48). In anotherembodiment of this aspect, X₁ comprises LS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 49). In another embodiment ofthis aspect, X₁ comprises ELS WNLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 50). In another embodiment of this aspect,X₁ comprises LELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQID NO: 51). In another embodiment of this aspect, X₁ comprises DLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 52). Inanother embodiment of this aspect, X₁ comprises QDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 53). In anotherembodiment of this aspect, X₁ comprises SQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 54). In another embodiment ofthis aspect, X₁ comprises KSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 55). In another embodiment of this aspect,X₁ comprises LKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 56). In another embodiment of this aspect, X₁ comprisesRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:57). In another embodiment of this aspect, X₁ comprises Q RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 58). Inanother embodiment of this aspect, X₁ comprises QQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 59). In anotherembodiment of this aspect, X₁ comprises EQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 60). In anotherembodiment of this aspect, X₁ comprises AEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 61). In anotherembodiment of this aspect, X₁ comprises RAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 62). In anotherembodiment of this aspect, X₁ comprises LRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 63). In anotherembodiment of this aspect, X₁ comprises ELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 64). In anotherembodiment of this aspect, X₁ comprises EELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 65). In anotherembodiment of this aspect, X₁ comprises LEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 66). In anotherembodiment of this aspect, X₁ comprises ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 67). In anotherembodiment of this aspect, X₁ comprises Q ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 68). Inanother embodiment of this aspect, X₁ comprises SQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 69). Inanother embodiment of this aspect, X₁ comprises ISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:70). In another embodiment of this aspect, X₁ comprises QISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:71). In another embodiment of this aspect, X₁ comprises TQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:72). In another embodiment of this aspect, X₁ comprises STQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 73). In another embodiment of this aspect, X₁ comprisesQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 74). In another embodiment of this aspect,X₁ comprises SQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 75). In another embodiment ofthis aspect, X₁ comprises KSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 76). In anotherembodiment of this aspect, X₁ comprises QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 77). Inanother embodiment of this aspect, X₁ comprises A QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:78). In another embodiment of this aspect, X₁ comprises MA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 79). In another embodiment of this aspect, X₁ comprises QMAQKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 80). In another embodiment of this aspect,X₁ comprises DQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 81). In another embodiment ofthis aspect, X₁ comprises GDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 82). Inanother embodiment of this aspect, X₁ comprises HGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 83). In another embodiment of this aspect, X₁ comprisesHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 84). In another embodiment ofthis aspect, X₁ comprises SHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 85). Inanother embodiment of this aspect, X₁ comprises ESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 86). In another embodiment of this aspect, X₁ comprisesLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 87).

In another embodiment of this aspect, X₁ comprises N LESHHGDQMAQKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 88). In another embodiment of this aspect,X₁ comprises KN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 89). In anotherembodiment of this aspect, X₁ comprises SKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 90). In another embodiment of this aspect, X₁ comprises VSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 91). In another embodiment ofthis aspect, X₁ comprises QVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:92). In another embodiment of this aspect, X₁ comprises SQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 93). In another embodiment ofthis aspect, X₁ comprises VSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:94). In another embodiment of this aspect, X₁ comprises NVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 95). In another embodiment ofthis aspect, X₁ comprises RNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:96). In another embodiment of this aspect, X₁ comprises ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 97). In another embodiment ofthis aspect, X₁ comprises A ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:98). In another embodiment of this aspect, X₁ comprises RA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 99). In another embodiment ofthis aspect, X₁ comprises ERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 100). In another embodiment of this aspect, X₁ comprisesEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 101). In anotherembodiment of this aspect, X₁ comprises LEERA ARNVSQVSKN LESHHGDQMAQKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 102). In another embodiment of this aspect,X₁ comprises QLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:103). In another embodiment of this aspect, X₁ comprises KQLEERAARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 104). In anotherembodiment of this aspect, X₁ comprises LKQLEERA ARNVSQVSKN LESHHGDQMAQKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLERLREEVTKLRM ELQVS (SEQ ID NO: 105). In another embodiment of this aspect,X₁ comprises SLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:106). In another embodiment of this aspect, X₁ comprises QSLKQLEERAARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 107). In anotherembodiment of this aspect, X₁ comprises T QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 108). In another embodiment ofthis aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQV(SEQ ID NO: 109). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQ (SEQ ID NO: 110).

In another embodiment of this aspect, X₁ comprises TT QSLKQLEERAARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM EL (SEQ ID NO: 111). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM E (SEQ ID NO: 112). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM (SEQID NO: 113). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLR (SEQ ID NO: 114). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKL (SEQ ID NO: 115). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTK (SEQ IDNO: 116). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEASDLLER LREEVT (SEQ ID NO: 117). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEV (SEQ ID NO: 118). In another embodiment of this aspect,X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREE (SEQ ID NO: 119). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LRE (SEQ ID NO: 120). In another embodiment of this aspect,X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LR (SEQ ID NO: 121). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER L (SEQ ID NO: 122). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER (SEQ ID NO: 123). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLE (SEQ ID NO: 124). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLL (SEQ ID NO: 125). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDL (SEQ ID NO: 126). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASD (SEQ ID NO: 127). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEAS(SEQ ID NO: 128). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE RNEA (SEQ ID NO: 129). In another embodiment ofthis aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNE (SEQ ID NO: 130). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RN(SEQ ID NO: 131). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQELNE R (SEQ ID NO: 132). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE (SEQ ID NO: 133). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELN (SEQ IDNO: 134). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKSQEL (SEQ ID NO: 135). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQE (SEQ ID NO: 136). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQ (SEQ IDNO: 137). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL SSFKS (SEQ ID NO: 138). In another embodiment of this aspect,X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFK (SEQ ID NO: 139). In another embodiment ofthis aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSF (SEQ ID NO: 140). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SS (SEQ ID NO:141). In another embodiment of this aspect, X₁ comprises TT QSLKQLEERAARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL S (SEQID NO: 142). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELSWNLNGLQADL (SEQ ID NO: 143). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQAD (SEQ ID NO: 144). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQA (SEQ ID NO: 145). In another embodimentof this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMAQKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQ (SEQ ID NO: 146). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGL (SEQ ID NO: 147). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNG (SEQ ID NO: 148). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLN (SEQ ID NO: 149). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNL (SEQ ID NO: 150). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WN (SEQ ID NO: 151). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS W (SEQ ID NO: 152). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS (SEQ ID NO: 153). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLEL (SEQ ID NO: 154). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLE (SEQ ID NO: 155). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDL (SEQ ID NO: 156). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQD (SEQ ID NO: 157). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQ (SEQ ID NO: 158). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKS (SEQ ID NO: 159). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLK (SEQ ID NO: 160). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RL (SEQ ID NO: 161). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ R (SEQ ID NO: 162). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ (SEQ ID NO: 163). In another embodimentof this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMAQKSQSTQISQ ELEELRAEQ (SEQ ID NO: 164). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAE (SEQ ID NO: 165). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRA (SEQ IDNO: 166). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELR (SEQ ID NO: 167). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEEL (SEQ ID NO: 168). In another embodiment ofthis aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEE (SEQ ID NO: 169). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELE (SEQ ID NO:170). In another embodiment of this aspect, X₁ comprises TT QSLKQLEERAARNVSQVSKN LESHHGDQMA QKSQSTQISQ EL (SEQ ID NO: 171). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ E (SEQ ID NO: 172). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ (SEQID NO: 173). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQIS (SEQ ID NO: 174). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQI (SEQ ID NO: 175). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQ (SEQ IDNO: 176). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQST (SEQ ID NO: 177). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQS (SEQ ID NO: 178). In another embodiment of this aspect,X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQ (SEQ ID NO: 179).In another embodiment of this aspect, X₁ comprises TT QSLKQLEERAARNVSQVSKN LESHHGDQMA QKS (SEQ ID NO: 180). In another embodiment ofthis aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QK (SEQ IDNO: 181). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA Q (SEQ ID NO: 182). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA (SEQ ID NO: 183). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA Q (SEQ ID NO: 184). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQM (SEQ ID NO: 185). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA Q (SEQ ID NO: 186). Inanother embodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQM (SEQ ID NO: 187). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN LESHHGDQ (SEQ ID NO: 188). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHGD(SEQ ID NO: 189). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA Q (SEQ ID NO: 190). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHHG(SEQ ID NO: 191). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESHHGDQMA Q (SEQ ID NO: 192). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LESHH(SEQ ID NO: 193). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSKN LESH (SEQ ID NO: 194). In another embodiment ofthis aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN LES (SEQ ID NO: 195).In another embodiment of this aspect, X₁ comprises TT QSLKQLEERAARNVSQVSKN LE (SEQ ID NO: 196). In another embodiment of this aspect, X₁comprises TT QSLKQLEERA ARNVSQVSKN L (SEQ ID NO: 197). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQVSKN (SEQ IDNO: 198). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQVSK (SEQ ID NO: 199). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVSQVS (SEQ ID NO: 200). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNVSQV (SEQ IDNO: 201). In another embodiment of this aspect, X₁ comprises TTQSLKQLEERA ARNVSQ (SEQ ID NO: 202). In another embodiment of thisaspect, X₁ comprises TT QSLKQLEERA ARNVS (SEQ ID NO: 203). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEERA ARNV (SEQ ID NO:204). In another embodiment of this aspect, X₁ comprises TT QSLKQLEERAARN (SEQ ID NO: 205). In another embodiment of this aspect, X₁ comprisesTT QSLKQLEERA AR (SEQ ID NO: 206). In another embodiment of this aspect,X₁ comprises TT QSLKQLEERA A (SEQ ID NO: 207). In another embodiment ofthis aspect, X₁ comprises TT QSLKQLEERA (SEQ ID NO: 208). In anotherembodiment of this aspect, X₁ comprises TT QSLKQLEER (SEQ ID NO: 209).

In some embodiments, conservative amino acid substitutions as set forthin Table 1 are permitted.

In another aspect, the compound has a formula of R₀—X₂, wherein R₀comprises SEQ ID No. 4 (or a derivative thereof such as e.g., SEQ ID No.4 having a D107E mutation) and X₂ comprises SEGSAE (SEQ ID NO: 9). Inone embodiment of this aspect, X₂ comprises the amino acid sequence AE.In another embodiment of this aspect, X₂ comprises EGSAE (SEQ ID NO:210). In another embodiment of this aspect, X₂ comprises GSAE (SEQ IDNO: 211). In another embodiment of this aspect, X₂ comprises SAE. Inanother embodiment of this aspect, X₂ comprises E. In other embodimentsof this aspect, X₂ can be —CONH₂, —COOH, NH₂, C₁-C₁₀ alkylamino,di(C₁-C₁₀ alkyl)amino, amino-C₁-C₁₀ alkylamino, or di(amino C₁-C₁₀alkyl)amino, NH(CH₂)NH wherein n is 1 to 8, or —OH. In anotherembodiment of this aspect.

In another embodiment of the aspects described above, the D at position107 of R₀ (SEQ ID No. 4) is modified to an E. In other embodiments ofthe aspects described above, the compounds comprise at least oneadditional R_(n) group (e.g., R₁, R₂, R₃, R₄, R₅, R₆ etc) between X₁ andR₀ or between X₂ and R₀, wherein R_(n) comprises any natural L-aminoacid, Pal, αNal, βNal, DpCl, CHx, where CHx is cyclohexyl, CHxAla or anyof their respective D-isomers; a halogen, diisobutylamide,dipropylamide, butylamide, pentylamide, dipentylamide, DPro, DPro-DPro,DVal, or DTrp. In one embodiment, the compound has the formula X₁—R₀—X₂,wherein X₁, R₀, and X₂ are as described above. X₁ and X₂ can haveconservative amino acid substitutions as set forth in Table 1. R₀ canalso have the substitutions set forth in Table 1. The compound can alsohave the formula R₀. In one embodiment, the compound comprises theformula R₀ with an amino acid substitution at position 107 of SEQ IDNo:4/ position 258 of sCD23 (SEQ ID No.3), for example the polypeptideof formula SEQ ID No:2. The D residue can be substituted for any naturalL-amino acid, a D-amino acid, or amino acid analogue as known in theart. In one embodiment, the D residue at position 258 of SEQ ID No.3(highlighted residue) is substituted with an E residue. As disclosed,infra, these compounds can be PEGylated.

Also contemplated herein are conservative amino acid substitutions ofthe sCD23 peptide or polypeptide. The terminology “conservative aminoacid substitutions” is well known in the art, which relates tosubstitution of a particular amino acid by one having a similarcharacteristic (e.g., similar charge or hydrophobicity, similarbulkiness). Examples include aspartic acid for glutamic acid, orisoleucine for leucine. A list of exemplary conservative amino acidsubstitutions is given in the table below. A conservative substitutionmutant or variant will 1) have only conservative amino acidsubstitutions relative to the parent sequence, 2) will have at least 90%sequence identity with respect to the parent sequence, preferably atleast 95% identity, 96% identity, 97% identity, 98% identity or 99% orgreater identity; and 3) will retain sCD23 activity (e.g., IgE bindingactivity) as that term is defined herein.

TABLE 1 CONSERVATIVE AMINO ACID SUBSTITUTIONS For Amino Acid CodeReplace With Alanine A D-ala, Gly, Aib, β-Ala, Acp, L-Cys, D-CysArginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D- Met,D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-GlnAspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine CD-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn,Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, Aib, β-Ala, Acp IsoleucineI D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg,D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-OrnMethionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp,Trans- 3,4 or 5-phenylproline, AdaA, AdaG, cis-3,4 or 5- phenylproline,Bpa, D-Bpa Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid,D-or-L-1- oxazolidine-4-carboxylic acid (Kauer, U.S. Pat. No.(4,511,390) Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met (O),D-Met (O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met,D-Met, Met (O), D-Met (O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe,L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met,AdaA, AdaG

An sCD23 peptide, polypeptide, or variant or derivative thereof can beproduced chemically by e.g., solution or solid-phase peptide synthesis,or semi-synthesis in solution beginning with protein fragments coupledthrough conventional solution methods, as described by Dugas et al(1981). Alternatively, an sCD23 peptide or polypeptide can besynthesized using e.g., recombinant methods.

A sCD23 peptide or polypeptide can be chemically synthesized, forexample, by the solid phase peptide synthesis of Merrifield et al(1964). Alternatively, a peptide or polypeptide can be synthesized usingstandard solution methods (see, for example, Bodanszky, 1984). Newlysynthesized peptides can be purified, for example, by high performanceliquid chromatography (HPLC), and can be characterized using, forexample, mass spectrometry or amino acid sequence analysis.

In one embodiment, the sCD23 peptide or polypeptide is producedrecombinantly. Systems for cloning and expressing polypeptides usefulwith the methods and compositions described herein include variousmicroorganisms and cells that are well known in recombinant technology.These include, for example, various strains of E. coli, Bacillus,Streptomyces, and Saccharomyces, as well as mammalian, yeast and insectcells. An sCD23 peptide or polypeptide can be produced as a peptide orfusion protein. Suitable vectors for producing peptides and polypeptidesare known and available from private and public laboratories anddepositories and from commercial vendors. See Sambrook et al, (1989).Recipient cells capable of expressing the gene product are thentransfected. The transfected recipient cells are cultured underconditions that permit expression of the recombinant gene products,which are recovered from the culture. Host mammalian cells, such asChinese Hamster ovary cells (CHO) or COS-1 cells, can be used. Thesehosts can be used in connection with poxvirus vectors, such as vacciniaor swinepox. Suitable non-pathogenic viruses that can be engineered tocarry the synthetic gene into the cells of the host include poxviruses,such as vaccinia, adenovirus, retroviruses and the like. A number ofsuch non-pathogenic viruses are commonly used for human gene therapy,and as carrier for other vaccine agents, and are known and selectable byone of skill in the art. The selection of other suitable host cells andmethods for transformation, culture, amplification, screening andproduct production and purification can be performed by one of skill inthe art by reference to known techniques (see, e.g., Gething et al,1981).

Native CD23 is a homotrimer, which permits high affinity binding of IgE.In some embodiments it is contemplated herein that a sCD23 compound usedherein is a homodimer, homotrimer, homo-multimer, a hetero-dimer, ahetero-trimer, or a hetero-multimer.

In other embodiments, the sCD23 compound used herein is a monomer.

In one embodiment a sCD23 peptide or polypeptide is isolated and/orpurified. Protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, thehomogenization and crude fractionation of the cells, tissue or organ topolypeptide and non-polypeptide fractions. The sCD23 peptide orpolypeptide of interest may be further purified using chromatographicand electrophoretic techniques to achieve partial or completepurification (or purification to homogeneity). Analytical methodsparticularly suited to the preparation of a pure peptide or polypeptideare ion-exchange chromatography, gel exclusion chromatography,polyacrylamide gel electrophoresis, affinity chromatography,immunoaffinity chromatography and isoelectric focusing. An example ofreceptor protein purification by affinity chromatography is disclosed inU.S. Pat. No. 5,206,347, the entire text of which is incorporated hereinby reference. A particularly efficient method of purifyingpeptides/polypeptides is fast performance liquid chromatography (FPLC)or even high performance liquid chromatography (HPLC).

A purified sCD23 peptide/polypeptide is intended to refer to acomposition, isolatable from other components, wherein the sCD23peptide/polypeptide is purified to any degree relative to the organismproducing recombinant protein or in its naturally-obtainable state. Anisolated or purified peptide or polypeptide, therefore, also refers to apeptide/polypeptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a sCD23 peptide/polypeptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which the sCD23peptide/polypeptide forms the major component of the composition, suchas constituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various methods for quantifying the degree of purification of a sCD23peptide/polypeptide are known to those of skill in the art and include,for example, determining the specific activity of an active fraction, orassessing the amount of polypeptides within a fraction by SDS/PAGEanalysis

Various techniques suitable for use in protein purification are known tothose of skill in the art and include, for example, precipitation withammonium sulfate, polyethylene glycol (PEG), antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxyapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the sCD23 peptide or polypeptide beprovided in the most purified state. Indeed, it is contemplated thatless purified products will have utility in certain embodiments. Partialpurification may be accomplished by using fewer purification steps incombination, or by utilizing different forms of the same generalpurification scheme. For example, it is appreciated that acation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculeto which it can specifically bind (e.g., a receptor-ligand interaction).The column material is synthesized by covalently coupling one of thebinding partners to an insoluble matrix. The column material is thenable to specifically adsorb the substance from the solution. Elutionoccurs by changing the conditions to those in which binding will notoccur (e.g., altered pH, ionic strength, temperature, etc.). The matrixshould be a substance that itself does not adsorb molecules to anysignificant extent and that has a broad range of chemical, physical andthermal stability. The ligand should be coupled in such a way as to notaffect its binding properties. The ligand should also provide relativelytight binding And it should be possible to elute the substance withoutdestroying the sample or the ligand.

Peptides, Polypeptides and Modifications Thereof

A sCD23 peptide/polypeptide functional to sequester IgE can beadministered directly to a subject in need thereof. In one approach, asCD23 peptide or polypeptide, produced, for example, in cultured cellsbearing a recombinant sCD23 expression vector can be administered to thesubject. The sCD23 compound will generally be administered intravenouslyor directly into the site of excess IgE in a subject e.g., byinhalation. This approach rapidly delivers the protein to the lungs andmaximizes the chance that the protein is intact when delivered. Furtheroptions for the delivery of sCD23 peptides/polypeptides as describedherein are discussed in the section “Pharmaceutical Compositions” hereinbelow.

In one embodiment, the protein or fragment thereof is linked to acarrier to enhance its bioavailability. Such carriers are known in theart and include poly(alkyl)glycol such as poly ethylene glycol (PEG).Fusion to serum albumin can also increase the serum half-life oftherapeutic polypeptides.

Vectors for transduction of a sCD23-encoding sequence are well known inthe art. While overexpression using a strong non-specific promoter, suchas a CMV promoter, can be used, it can be helpful to include a tissue-or cell-type-specific promoter on the expression construct for example,the use of an immune cell-specific promoter (e.g., interleukin promoteror TNF-α promoter) or other cell-type-specific promoter can beadvantageous, depending upon what cell type is used as a host. Further,treatment can include the administration of viral vectors that drive theexpression of sCD23 peptides or polypeptides in infected host cells.Viral vectors are well known to those skilled in the art.

The vectors are readily adapted for use in the methods of the presentinvention. By the appropriate manipulation using recombinantDNA/molecular biology techniques to insert an operatively linkedsCD23-encoding nucleic acid segment into the selectedexpression/delivery vector, many equivalent vectors for the practice ofthe methods described herein can be generated. It will be appreciated bythose of skill in the art that cloned genes readily can be manipulatedto alter the amino acid sequence of a protein.

The sCD23 peptide or polypeptide can also be a fusionpeptide/polypeptide, fused, for example, to a polypeptide that targetsthe product to a desired location, or, for example, a tag thatfacilitates its purification, if so desired. Fusion to a polypeptidesequence that increases the stability of the sCD23 compound is alsocontemplated. For example, fusion to a serum protein, e.g., serumalbumin, can increase the circulating half-life of a sCD23 peptide orpolypeptide. Tags and fusion partners can be designed to be cleavable,if so desired. Another modification specifically contemplated isattachment, e.g., covalent attachment, to a polymer. In one aspect,polymers such as polyethylene glycol (PEG) or methoxypolyethylene glycol(mPEG) can increase the in vivo half-life of proteins to which they areconjugated. Methods of PEGylation of polypeptide agents are well knownto those skilled in the art, as are considerations of, for example, howlarge a PEG polymer to use. In another aspect, biodegradable orabsorbable polymers can provide extended, often localized, release ofpolypeptide agents. Such synthetic bioabsorbable, biocompatiblepolymers, which may release proteins over several weeks or months caninclude, for example, poly-α-hydroxy acids (e.g. polylactides,polyglycolides and their copolymers), polyanhydrides, polyorthoesters,segmented block copolymers of polyethylene glycol and polybutyleneterephtalate (POLYACTIVE™), tyrosine derivative polymers orpoly(ester-amides). Suitable bioabsorbable polymers to be used inmanufacturing of drug delivery materials and implants are discussed e.g.in U.S. Pat. Nos. 4,968,317; 5,618,563, among others, and in “BiomedicalPolymers” edited by S. W. Shalaby, Carl Hanser Verlag, Munich, Vienna,New York, 1994 and in many references cited in the above publications.The particular bioabsorbable polymer that should be selected will dependupon the particular subject that is being treated.

In one embodiment, the sCD23 peptide/polypeptide is modified to includesalts and/or chemical derivatives. As used herein, the term “chemicalderivative” refers to a sCD23 peptide or polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules can include, for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. Also included aschemical derivatives are sCD23 peptides or polypeptides that contain oneor more naturally occurring amino acid derivatives of the twentystandard amino acids. For example, 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

In another embodiment, the sCD23 peptide or polypeptide is modified toincrease the stability in solution and, therefore, serve to prolong thehalf-life of the peptide/polypeptide inhibitor in solutions,particularly biological fluids, such as blood, plasma or serum, byblocking proteolytic activity in the blood. A sCD23 peptide/polypeptidecan have a stabilizing group at one or both termini including, forexample, amido, acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkylcarbonyl, benzyloxycarbonyl and the like end group modifications.Additional modifications include using a “L” amino acid in place of a“D” amino acid at the termini, cyclization of the peptide inhibitor, andamide rather than amino or carboxy termini to inhibit exopeptidaseactivity.

A sCD23 peptide/polypeptide as used herein may or may not beglycosylated. sCD23 peptides/polypeptides are not glycosylated, forexample, when produced directly by synthesis techniques or are producedin a prokaryotic cell transformed with a recombinant polynucleotide.Peptide or polypeptide molecules produced in eukaryotic expressionsystems (such as, for example, Saccharomyces cerevisiae-based expressionsystems, baculovirus-based expression systems utilizing for example, Sf9insect cells, and mammalian expression systems) are typicallyglycosylated.

In one embodiment, the sCD23 peptide/polypeptide is maintained in aconstrained secondary conformation. The terms “constrained secondarystructure,” “stabilized” and “conformationally stabilized” indicate thatthe bonds comprising the peptide or polypeptide are not able to rotatefreely but instead are maintained in a relatively fixed structure. Amethod for constraining the secondary structure of a newly synthesizedlinear peptide is to cyclize the peptide using any of various methodswell known in the art. For example, a cyclized sCD23 peptide can beprepared by forming a peptide bond between non-adjacent amino acidresidues as described, for example, by Schiller et al (1985). Peptidescan be synthesized on the Merrifield resin by assembling the linearpeptide chain using Nα-Fmoc-amino acids and Boc and tertiary-butylproteins. Following the release of the peptide from the resin, a peptidebond can be formed between the amino and carboxy termini.

A newly synthesized linear peptide or polypeptide can also be cyclizedby the formation of a bond between reactive amino acid side chains. Forexample, a peptide containing a cysteine-pair can be synthesized, with adisulfide bridge, can be formed by oxidizing a dilute aqueous solutionof the peptide with K₃Fe(CN)₆. Alternatively, a lactam such as anε-(γ-glutamyl)-lysine bond can be formed between lysine and glutamicacid residues, a lysinonorleucine bond can be formed between lysine andleucine residues or a dityrosine bond can be formed between two tyrosineresidues. Cyclic peptides can be constructed to contain, for example,four lysine residues, which can form the heterocyclic structure ofdesmosine (see, for example, Devlin, 1997). Methods for forming theseand other bonds are well known in the art and are based on well-knownrules of chemical reactivity (Morrison et al, 1992).

In one embodiment, the sCD23 peptide or polypeptide (e.g., isolated,synthetic, or recombinant peptide) is attached to, or enclosed orenveloped by, a macromolecular complex. The macromolecular complex canbe, without limitation, a virus, a bacteriophage, a bacterium, aliposome, a microparticle, a targeting sequence, a nanoparticle (e.g., agold nanoparticle), a magnetic bead, a yeast cell, a mammalian cell, acell or a microdevice. These are representative examples only andmacromolecular complexes within the scope of the methods andcompositions described herein can include virtually any complex that canattach or enclose a peptide/polypeptide and be administered to asubject.

If desired, the isolated sCD23 peptide/polypeptide can be attached to asolid support, such as, for example, magnetic beads, Sepharose beads,agarose beads, a nitrocellulose membrane, a nylon membrane, a columnchromatography matrix, a high performance liquid chromatography (HPLC)matrix or a fast performance liquid chromatography (FPLC) matrix forpurification. In one embodiment, the isolated sCD23 peptide/polypeptidecan be attached to a scaffold or other device for local and/or sustaineddelivery of the sCD23 peptide to a site of allergic response.

In one embodiment, the sCD23 peptide/polypeptide comprises a fusionprotein. These molecules generally have all or a substantial portion ofthe sCD23 peptide, linked at the N- or C-terminus, to all or a portionof a second polypeptide or protein. For example, fusions may employleader sequences from other species to permit the recombinant expressionof a protein in a heterologous host. Another useful fusion includes theaddition of an immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as, for example, activesites from enzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

The fusion proteins described herein can comprise a sCD23 peptide orpolypeptide linked to a second therapeutic protein or peptide. Examplesof proteins or peptides that may be incorporated into a fusion proteininclude, but are not limited to, cytostatic proteins, cytocidalproteins, pro-apoptosis agents, anti-angiogenic agents, hormones,cytokines, growth factors, peptide drugs, antibodies, Fab fragmentsantibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins,cell adhesion proteins and binding proteins. These examples are notmeant to be limiting and it is contemplated herein that virtually anyprotein or peptide known to one of skill in the art could beincorporated into a fusion protein comprising a targeting peptide.Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion protein, or by attachment of a DNAsequence encoding the targeting peptide to a DNA sequence encoding thesecond peptide or protein, followed by expression of the intact fusionprotein.

Protein PEGylation

Polyethylene glycol (PEG) can be conjugated to the peptide orpolypeptide compounds as described herein. PEGylation can be achieved byincubation of a reactive derivative of PEG with the targetmacromolecule. The conjugation to PEG can be performed eitherenzymatically or chemically, the methods of which are well establishedin the art (Chapman, 2002; Veronese and Pasut, 2005). With PEGylationthe total size of a peptide can be increased, which reduces the chanceof renal filtration and can increase the circulating half-life of thepeptide. PEGylation further protects peptides from proteolyticdegradation and slows the clearance from the blood. In addition,PEGylation reduces immunogenicity and increases solubility ofmacromolecules (e.g., peptides). The improved pharmacokinetics by theaddition of PEG is due to several different mechanisms: increase in sizeof the molecule, protection from proteolysis, reduced antigenicity, andthe masking of specific sequences from cellular receptors. For example,in the case of antibody fragments (Fab), a 20-fold increase in plasmahalf-life has been achieved by PEGylation (Chapman, 2002).

PEG moieties useful with the compositions and methods described hereininclude PEG polymers, derivatives and PEG lipids. PEG polymers can bee.g., linear, branched or multi-armed, among others. The PEG conjugateaccording to the present invention may be of any molecular weight, forexample, the molecular weight may be between 500 and 100,000 Da, between500 and 60,000 Da, between 1000 and 40,000 Da, or between 5000 and40,000 Da. PEGs having molecular weights of 10000 Da, 20000 Da, 30000 Daor 40000 Da may be used with the peptides or polypeptides describedherein.

In addition, other polymers are also contemplated for use with themethods and compositions described herein and include, but are notlimited to, poly(alkylene glycols) such as poly(propylene glycol)(“PPG”), copolymers of ethylene glycol and propylene glycol and thelike, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(.alpha.-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), and combinations of any of the foregoing.Representative polymeric reagents and methods for conjugating thesepolymers to an active moiety are known in the art and further describedin Zalipsky, S., et al., “Use of Functionalized Poly(Ethylene Glycols)for Modification of Polypeptides” in Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press,New York (1992), and in Zalipsky (1995) Advanced Drug Reviews 6:157-182.

Dosage and Administration

In one aspect, the methods described herein provide a method fortreating an IgE mediated disease in a subject. In one embodiment, thesubject can be a mammal. In another embodiment, the mammal can be ahuman, although the approach is effective with respect to all mammals.The method comprises administering to the subject an effective amount ofa pharmaceutical composition comprising a sCD23 peptide/polypeptide, ina pharmaceutically acceptable carrier.

The dosage range for the sCD23 compound depends upon the potency androute of administration, and include amounts large enough to produce thedesired effect, e.g., a measurable decrease in at least one symptom ofan IgE mediated disease such as e.g., an allergic response. The dosageshould not be so large as to cause unacceptable adverse side effects.Generally, the dosage will vary with the particular compound used andwith the age, condition, and sex of the patient. The dosage can bedetermined by one of skill in the art and can also be adjusted by theindividual physician in the event of any complication. Typically, thedosage ranges from 0.001 mg/kg body weight to 5 g/kg body weight. Insome embodiments, the dosage range is from 0.001 mg/kg body weight to 1g/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kg body weight,from 0.001 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kgbody weight to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight,from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001 mg/kgbody weight to 1 mg/kg body weight, from 0.001 mg/kg body weight to 0.1mg/kg body weight, from 0.001 mg/kg body weight to 0.005 mg/kg bodyweight. Alternatively, in some embodiments the dosage range is from 0.1g/kg body weight to 5 g/kg body weight, from 0.5 g/kg body weight to 5g/kg body weight, from 1 g/kg body weight to 5 g/kg body weight, from1.5 g/kg body weight to 5 g/kg body weight, from 2 g/kg body weight to 5g/kg body weight, from 2.5 g/kg body weight to 5 g/kg body weight, from3 g/kg body weight to 5 g/kg body weight, from 3.5 g/kg body weight to 5g/kg body weight, from 4 g/kg body weight to 5 g/kg body weight, from4.5 g/kg body weight to 5 g/kg body weight, from 4.8 g/kg body weight to5 g/kg body weight. In one embodiment, the dose range is from 5 m/kgbody weight to 30 m/kg body weight. Alternatively, the dose range willbe titrated to maintain serum levels between 5 μg/mL and 30 m/mL.

In one embodiment, a sCD23 peptide/polypeptide as described herein isadministered directly to a site of an allergic response by e.g.,injection, inhalation, polymer or scaffold-mediated delivery etc.

Administration of the doses recited above can be repeated, if necessary,for a limited period of time. In some embodiments, the doses are givenonce a day, or multiple times a day, for example but not limited tothree times a day. In a preferred embodiment, the doses recited aboveare administered daily for several weeks or months. The duration oftreatment depends upon the subject's clinical progress andresponsiveness to therapy. Continuous, relatively low maintenance dosesare contemplated after an initial higher therapeutic dose.

A therapeutically effective amount is an amount of an agent that issufficient to produce a statistically significant, measurable change inat least one symptom of an inflammatory disease etc. (see “EfficacyMeasurement” below). Such effective amounts can be gauged in clinicaltrials as well as animal studies for a given sCD23 peptide orpolypeptide.

Agents useful in the methods and compositions described herein can beadministered by e.g., inhalation, topically, direct injection,intravenously (by bolus or continuous infusion), orally,intraperitoneally, intracavity, and can be delivered by peristalticmeans, if desired, or by other means known by those skilled in the art.In one embodiment, the compositions are delivered by means of ascaffold, polymer, gel etc. for local delivery of sCD23 peptide.

Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose. The term “unit dose” whenused in reference to a therapeutic composition refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredphysiologically acceptable diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. An agent can be targeted by meansof a targeting moiety, such as e.g., an antibody or targeted liposometechnology. In some embodiments, a sCD23 peptide is targeted to aspecific tissue by using bispecific antibodies, for example produced bychemical linkage of an anti-ligand antibody (Ab) and an Ab directedtoward a specific target. To avoid the limitations of chemicalconjugates, molecular conjugates of antibodies can be used forproduction of recombinant bispecific single-chain Abs directing ligandsand/or chimeric inhibitors at cell surface molecules. The addition of anantibody to a sCD23 peptide or polypeptide permits the agent attached toaccumulate additively at the desired target site. Antibody-based ornon-antibody-based targeting moieties can be employed to deliver aligand or the inhibitor to a target site. Preferably, a natural bindingagent for an unregulated or disease associated antigen is used for thispurpose.

Pharmaceutical Compositions

The present invention involves therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions contain a physiologically tolerable carrier together with asCD23 peptide or polypeptide as described herein, dissolved or dispersedtherein as an active ingredient. In a preferred embodiment, thetherapeutic composition is not immunogenic when administered to a mammalor human patient for therapeutic purposes, unless so desired. As usedherein, the terms “pharmaceutically acceptable”, “physiologicallytolerable” and grammatical variations thereof, as they refer tocompositions, carriers, diluents and reagents, are used interchangeablyand represent that the materials are capable of administration to orupon a mammal without the production of undesirable physiologicaleffects such as nausea, dizziness, gastric upset and the like. Apharmaceutically acceptable carrier will not promote the raising of animmune response to an agent with which it is admixed, unless so desired.The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein.

Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like and combinations thereof. In addition, if desired,the composition can contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient. The therapeuticcomposition of the present invention can include pharmaceuticallyacceptable salts of the components therein. Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Physiologically tolerablecarriers are well known in the art. Exemplary liquid carriers aresterile aqueous solutions that contain no materials in addition to theactive ingredients and water, or contain a buffer such as sodiumphosphate at physiological pH value, physiological saline or both, suchas phosphate-buffered saline. Still further, aqueous carriers cancontain more than one buffer salt, as well as salts such as sodium andpotassium chlorides, dextrose, polyethylene glycol and other solutes.Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active compound used in the invention thatwill be effective in the treatment of a particular disorder or conditionwill depend on the nature of the disorder or condition, and can bedetermined by standard clinical techniques.

Monitoring Efficacy of Treatment

The efficacy of a given treatment for an IgE mediated disease can bedetermined by the skilled clinician. However, a treatment is considered“effective treatment,” as the term is used herein, if any one or more ofthe signs or symptoms of an IgE mediated disease, for example, allergicresponses such as e.g., coughing, sneezing, mucus production, rhinitis,itchy eyes, anaphylactic response to allergen, skin irritation, redness,inflammation, breathing difficulties etc. are altered in a beneficialmanner, other clinically accepted symptoms or markers of disease areimproved, or ameliorated. In one embodiment, the improvement is seen asa need for fewer anti-allergy treatments (e.g., allergy shots, steroids,etc), fewer episodes of hospitalization, and/or longer intervals betweenhospitalizations, than the individual has experienced prior to treatmentwith the peptide/polypeptide. Efficacy can also be measured by a failureof an individual to worsen as assessed by hospitalization or need formedical interventions (i.e., progression of the disease is halted or atleast slowed). Methods of measuring these indicators are known to thoseof skill in the art and/or described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human, or a mammal) and includes: (1) inhibiting thedisease, e.g., arresting, or slowing progress of an IgE mediated diseaseor reaction; or (2) relieving the disease, e.g., causing regression ofsymptoms. The methods can also be used to prevent or reduce thelikelihood of the development of a chronic condition (e.g., asthma,eczema) or complication relating to an IgE mediated disease.

An effective amount for the treatment of an IgE mediated disease meansthat amount which, when administered to a mammal in need thereof, issufficient to result in effective treatment as that term is definedherein. Efficacy of a peptide/polypeptide compound can be determined byassessing physical indicators of an IgE mediated disease, for example,congestion, coughing, sneezing, redness, itchiness, anaphylaxis,wheezing, swelling, etc.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

EXAMPLES

The inventors have identified an important molecule present in worminfected-people that is involved in modulating the immune system in amanner that prevents allergic symptoms. These findings indicate thatthis molecule works by interfering with IgE, a component of the immunesystem that initiates most allergic reactions including sneezing,coughing, sinus congestion, mucus production in the sinuses (rhinitis)or lungs (asthma).

Example 1 Background

People Infected with Parasitic Worms Lack Bona Fide Allergic Responses

Parasitic worm infection is classically associated with high levels ofcirculating eosinophils, activated mast cells and IgE, the majorcomponents that mediate allergic responses. Paradoxically, peopleinfected with parasitic worms, do not demonstrate clinical allergicsymptoms. A portion of this has been attributed to the “hygienehypothesis” that states a lack of exposure to parasites increasessusceptibility to allergic and other chronic inflammatory diseases.Thus, the basis of the hypothesis stems from the idea that wormsimmuno-modulate the immune system in a manner that prevents exaggeratedor inappropriate immune responses to innocuous antigens, such as pollenor nut proteins. For example, inflammatory bowel disease is believed tostem from an unregulated immune response to normally non-pathogeniccommensal microbes in the gut. In fact, ulcerative colitis patients canbe cured of their disease through the treatment of parasitic worms.

Inflammatory bowel disease and other diseases are thought to be mediatedby the opposite arm of the immune system that causes allergy. However,allergy is mediated by the very same mediators that respond to worminfection Animal models have demonstrated that certain cytokines mightbe upregulated in response to certain worm antigens that might attenuatethe allergic response or development of the response to innocuousantigens.

The methods and compositions described herein offer a compelling linkbetween the prevention of allergic responses and worm infection. Thepreliminary data demonstrate that schistosomes, a parasitic flat wormthat infects over 207 million people worldwide, modifies a host proteinin a specific manner that leads to the prevention of classic allergicresponses. Thus the data described herein indicate that schistosomesproduce a protease that cleaves CD23, the low affinity IgE receptor, ina specific manner. The resulting cleaved soluble (sCD23) product isdifferent than that naturally produced by the host and has not beenpreviously described.

Human CD23 is a cell bound IgE receptor that can be differentiallycleaved by host- and microbial-proteases. Cleaved proteins exist asactive soluble receptors and their effect depends upon their size andself-conjugation forms. sCD23 has multiple ligands including IgE, CD21and perhaps Mac-1. Thus the effect of the different sCD23 isoformsdepends upon the cell for which it binds to as well (FIG. 1). Themethods and compositions described herein are based, in part, onobservations in clinical samples and experimental data demonstratingthat a small ˜15 kDa sCD23 protein that binds IgE (FIG. 2) but not CD21reduces allergy and other IgE-mediated diseases.

The protein sequence of Modified sCD23 includes residues 156-292:

Modified sCD23 for the treatment of IgE-mediated disease (SEQ ID NO: 1)156 -SGFVC NTCPEKWINF QRKCYYFGKG TKQWVHARYACDDMEGQLVS IHSPEEQDFL TKHASHTGSW IGLRNLDLKGEFIWVDGSHV DYSNWAPGEP TSRSQGEDCV MMRGSGRWNDAFCDRKLGAW VCDRLATCTP PA -192 C TERMINUS

Example 2 Increasing IgE Binding by Changing a Heptavalent Ca++Chelation Site in CD23 to an Octavalent Chelation Site

CD23 is a calcium binding protein. Calcium is important for the bindingof CD23 to IgE and the mechanism of calcium binding has been described.CD23 bears significant sequence similarity to other calcium bindinglectins such as the human asialoglycoprotein receptor, mannose-bindingprotein (MBP), and DC-SIGN. The best characterized of these proteins,MBP is know to chelate calcium using 8 atoms to form a square pyramidalchelation structure. In contrast, CD23 appears to bind calcium usingonly 7 atoms. Because regions associated with calcium chelation are alsoassociated with IgE binding it is desirable for this region to be asstable as possible. To promote the stability of Ca++ binding, the CD23fragment is mutated such that it carries an additional carboxyl groupcapable of donating electron density. Without wishing to be bound bytheory, the hypothesis is that this will stabilize the L1 loop that isimportant for the binding of IgE, increasing its affinity for the ligandand decreasing any requirement for high calcium concentrations.

Residue 258 (an aspartic acid) is mutated to a glutamic acid. Thismutation will lead to the formation of an octavalent chelation site thatwill stabilize calcium binding and increase IgE binding affinity.

Modified sCD23 with higher IgE binding capacity(High Affinity) (underlined: D258 to E) SEQ ID NO: 2SGFVC NTCPEKWINF QRKCYYFGKG TKQWVHARYA CDDMEGQLVSIHSPEEQDFL TKHASHTGSW IGLRNLDLKG EFIWVDGSHV DYSNWAPGEP TSRSQGE ECV MMRGSGRWND AFCDRKLGAW VCDRLATCTP PA

This ˜15 kD protein is an advantageous treatment for allergic diseasesbecause it is found naturally in human blood and causes few sideeffects. Furthermore, mice express a similar protein that lacks the CD21binding site, which has been shown to negatively regulate IgEproduction.

Example 3 Polypeptide Variations

Modified sCD23 contains the lectin head which binds IgE but lacks theCD21 tail. The CD21-binding tail encompasses amino acids 290 to 321.Sm-sCD23 is a non-CD21 binding protein that lacks amino acids 293-321.CD21 complement binding requires at least amino acids 290-298.Variations of this protein include producing sCD23 lacking any aminoacids from 290 to 321.

SEQ ID No: 3 MEEGQYSEIE ELPRRRCCRR GTQIVLLGLV TAALWAGLLT LLLLWHWDTTQSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ

ELQVSSGFVC N TCPEKWINF QRKCYYFGKG TKQWVHARYA CDDMEGQLVSIHSPEEQDFL TKHASHTGSW IGLRNLDLKG EFIWVDGSHV DYSNWAPGEP

GPDSRPDPDG RLPTPSAPLH S underlined text = lectin IgE binding domainitalicized text = C-tail CD21 binding domain bold text =start of common sCD23 polypeptide (172 aa)

Sequence of Modified sCD23 (SEQ ID No: 4)SGFVC NTCPEKWINF QRKCYYFGKG TKQWVHARYACDDMEGQLVS IHSPEEQDFL TKHASHTGSW IGLRNLDLKGEFIWVDGSHV DYSNWAPGEP TSRSQGEDCV MMRGSGRWND AFCDRKLGAW VCDRLATCTP PAPolypeptide Variation A (SEQ ID NO: 5)A monomeric protein that retains IgE-bindingproperties but not CD21-binding abilityTT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS SGFVC NTCPEKWINF QRKCYYFGKG TKQWVHARYA CDDMEGQLVS IHSPEEQDFLTKHASHTGSW IGLRNLDLKG EFIWVDGSHV DYSNWAPGEPTSRSQGEDCV MMRGSGRWND AFCDRKLGAW VCDRLATCTP PA SEGSAEPolypeptide Variation B High Affinity Modified sCD23 (SEQ ID NO: 6)TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS SGFVC NTCPEKWINF QRKCYYFGKG TKQWVHARYA CDDMEGQLVS IHSPEEQDFLTKHASHTGSW IGLRNLDLKG EFIWVDGSHV DYSNWAPGEPTSRSQGEECV MMRGSGRWND AFCDRKLGAW VCDRLATCTP PA SEGSAE

Example 4 Advantages Over Current Treatments Avoid Anaphylactic Shockand Other Severe Side Effects

Modified sCD23 is a low-affinity IgE receptor and can only bind one IgEFc molecule. In contrast, Xolair is a monoclonal antibody which has 2binding sites and has the potential to cross-link IgE on the cellsurface (FIG. 5; ˜2% reported incidence). Therefore, it is unlikely thatmodified sCD23 will have the ability to induce anaphylactic shock thatis seen with the use of Xolair. Further, due to its potency, Xolair hasalso been associated with malignancy and Churg-Strauss syndrome, whichis an autoimmune vasculitis, as well.

Xolair® has already been shown to be effective in reducing allergicdisease, but is prohibitively expensive and potentially dangerous.However, modified sCD23, which has a similar mechanism of action asXolair, represents an ideal, low cost allergic treatment. In addition,anti-IgE mediated therapy has proven effective in other diseases such ascorticosteroid induced diabetes in bronchopulmonary aspergillosis andcystic fibrosis highlighting other potential uses for modified sCD23.

Reduce Health Care Costs Associated with Allergy

Modified sCD23 has a similar mechanism of action as Xolair (except forthe ability to induce anaphylaxis.) Xolair is prohibitively costly dueto its mechanism of action and may cause anaphylactic shock. Thereforethis treatment must be administered in a physician's office. It isunlikely that modified sCD23 will induce anaphylactic shock and thus therequirement for administration in a physician's office will be removed.Removal of this requirement opens the large market of the complex arrayof allergic treatments for a broad patient base. In general, patientsreport low satisfaction with allergy treatments. Intranasal corticoidsteroids (INCS) are considered the gold-standard for AR but thistreatment is associated with bone mineral lost, growth retardation, andadrenal suppression which is troublesome for children. Modified sCD23will not produce these side effects thereby reducing the cost of carefor patients with INCS-induced adverse effects.

Over the counter (OTC) anti-histamine/decongestants also have adverseeffects, such as drowsiness, epistaxis, and burning and have thetendency to stop working. In contrast, modified sCD23 is not likely toinduce these side-effects either. sCD23 is an effective treatment andpatients will reduce their cost of attempting to provide relief withmultiple over the counter drugs.

Treatment to a Broad Allergic Population

Currently, Xolair is only cost-effective for a small population ofasthmatic patients. Because of the predicted low production costs andincreased safety, modified sCD23 is an effective treatment for a broadrange of IgE-mediated diseases, including allergy. Further, due to itsnatural chemical nature, modified sCD23 can be used in children andpregnancy.

Market the Product as Safe and Effective

Modified sCD23 appears to be found in human sera at low concentrationsin people who are not infected with schistosomes. In contrast, modifiedsCD23 appears to be found in high concentrations in people who areinfected with schistosomes, highlighting the potential safety of thetreatment.

Example 5 Methods for Testing Binding Properties of a sCD23 Peptide orPolypeptide

The data provided herein indicate that incubation of CD23+ cells withschistosome antigens reduces surface CD23 levels. It has been shown thatmultiple host and microbe-derived proteases may differentially cleaveCD23, therefore the cell supernatants of B cells treated withschistosome antigens were evaluated to determine the characteristics ofthe cleaved products by western blot. Supernatants from B cells treatedwith schistosome antigens were enriched with a 15 kDa product (FIG. 2).As certain cleaved sCD23 peptides can bind both IgE and CD21, thebinding properties of the 15 kDa protein were tested. CD23 is known tointeract with CD21 on two sites of CD21. The reagent used in this assaywas rhuCD21 from XpressBio which is a 43 kDa protein aa: 130-280 (out of1092) and spans 1-2 of the short consensus repeats (SCR). This regioncontains the necessary binding for CD23 but it is not known how thismight affect the ability of CD23 to bind IgE. The intensity of the 15kDa band was decreased in the presence of sCD21 indicating that sCD21blocks cleavage of CD23 by schistosomes (FIG. 2B). Further, CD21 did notappear to co-precipitate with the 15 kDa sCD23. In contrast, FIG. 2demonstrates that IgE can be immuno-precipitated with sCD23 indicatingthat schistosome-generated sCD23 binds IgE. Thus, the CD21 binding siteon the tail is likely eliminated.

Without wishing to be bound by theory, sequestration of IgE byschistosome modified sCD23 may be an immuno-evasion tactic to promoteparasitism. It is hypothesized that promotion of parasitism may alsobenefit the host by preventing anaphylaxis and other severe allergicreactions in the presence of copious IgE and eosinophils.

Human soluble CD23 is modified by schistosomes (a protease cleavageevent), which produces a CD23 fragment that binds IgE, but not CD21.This novel CD23 fragment likely plays an important role in controllinginappropriate IgE mediated immunity in schistosomiasis and that it isthe IgE-only binding characteristic of this fragment that is important.A recombinant form of the schistosome-cleaved CD23 fragment is providedherein and can be epitope tagged to facilitate analysis.

His6 (SEQ ID NO: 12) and Myc epitope-tagged fragments of CD23 thatcontain amino acids 156-292 were chosen since existing data indicatesthat this fragment will fold properly and will contain the IgE bindingdomain but will lack the CD21 binding domain. The NMR structure of asoluble CD23 protein that binds both IgE and CD21 shows that each ofthese molecules binds at a structurally distinct place on the CD23molecule. IgE binds to the lectin-head region (including residues from(aa187 to 279), while CD21 appears to involve residues that areseparated in both primary structure and tertiary structure (residues 294and above). Thus, a fragment from 156-292 includes the lectin bindingregion that is important for binding to IgE but is devoid of theresidues required for CD21 interaction.

In designing the recombinant fragment it was elected to add two separatetypes of affinity tags. The His6 (SEQ ID NO: 12) sequence serves as anaffinity-tag for purification. The His-tag was chosen over other wellestablished protein-fusion strategies (i.e. schistosome GST, MaltoseBinding protein) because the His-tag adds minimally to the overallmolecular weight and structure of the protein fragment. The secondaffinity tag, the Myc epitope serves to provide a high-affinity antibodybinding region to allow biochemical (immuno-precipitation) andimmunological (FACS) manipulation and identification of the fusionprotein.

To achieve this, a small portion of CD23 is subcloned into the bacterialexpression vector pET 28a. This vector has the advantage of having aHis6 tag (SEQ ID NO: 12) designed into the start of the translatedprotein sequence. The CD23 156-292 fragment is placed into this vectorby using two primers. The first contains a NheI cleavage site and addsthe c-Myc epitope sequence EQKLISEEDL (SEQ ID NO: 212) 5′ to the CD23sequence. The second contains a stop codon and a HindIII site that arearranged so that they are 3′ to the codon for amino acid 292 of CD23.The codon bias for the c-Myc sequence is altered to optimize expressionin E. coli (for example, the sequence can be: GAA CAG AAA CTG ATC TCTGAA GAA GAC CTG (SEQ ID NO: 213)). CD23 is PCR amplified from total RNAfrom tonsil B cells using these primers and then the cDNA fragment isligated into pET28a (the CD23 fragment does not itself contain either anNheI or HindIII cleavage site so these will be unique to the primers).The resulting plasmid is sequenced to verify proper cloning, and theplasmid is then transformed into a protein expressing strain of E. coli(e.g. BLR pLysS or comparable DE3+ strain). Expression of the CD23fragment is optimized using standard procedures. The fragment can bepurified on Ni-NTA agarose and is removed from the agarose column eitherby imidazole washes or by cleavage of the His tag from the peptide usingthe thrombin protease.

Example 6 Methods for Testing Efficacy of Modified sCD23 to PreventAllergic Symptoms in Vivo

B6.Cg-Fcer1a^(tm1Knt) Tg(FCER1A)1Bhk/J which express the Fc portion ofhuman IgE and the human FcεRI α-chain, which binds IgE. These mice havebeen shown to respond to experimental induction of anaphylaxis and arean ideal model to test the efficacy of Modified sCD23 in preventingallergic reactions.

Mice are injected intravenously with 20 μg of human IgE specific for NP(Serotec). 24 hours later, mice are injected with NP-BSA to inducecross-linking of cell bound IgE and anaphylaxis-like disease. Evans bluedye is co-injected with NP-BSA to track vessel leakage into the tissues.

Mice can be pre-treated with 1, 10, 50, 100, or 200 μg of Modified sCD23two hours prior to the injection of IgE. 24 hours later, peripheralblood and tissue FcεR1+ cells are measured for the level of cell-boundIgE.

Once an appropriate dose of a particular peptide or polypeptide ischosen, mice are pre-treated with that concentration of Modified sCD23two hours prior to the injection of IgE. 24 hours later, NP-BSA isinjected into mice and symptoms of allergy are measured as belowcompared to mice that have received no IgE or no Modified sCD23.

Measurement of Allergy in Mice

Symptoms of systemic anaphylaxis appear within 15 to 30 minutes andreach a peak at 40 to 50 minutes after the first symptoms appear. Asymptom scoring system is used according to previously describedparameters of symptoms for determining IgE-mediated responses in mice.Briefly, 0 is assigned if no symptoms are evident, and 1 through 5 areassigned if symptoms are observed, where 1 represents mild scratching,rubbing, or both of the nose, head, or feet; 2 and 3 representintermediate symptoms (e.g., edema around the eyes or mouth, pilarerection, and/or labored breathing); 4 represents significantly reducedmotility, tremors, and/or significant respiratory distress; and 5represents death. One hour after challenge, mice are bled for plasmahistamine levels. Twenty-four hours later, mice are euthanized, andtissues collected for analysis.

Plasma histamine levels are determined by using an EIA kit from BectonDickenson (Franklin Lakes, N.J.), as per the manufacturer'sinstructions.

To detect vascular leakage footpads and ears of mice are examined forsigns of vascular leakage (visible blue color) 30 to 40 minutes afterdye/antigen administration.

1. (canceled)
 2. A compound of the formula X₁—R₀ (SEQ ID NO: 214),wherein R₀ comprises SEQ ID NO: 4 and X₁ comprises TT QSLKQLEERAARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADLSSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 7), or a contiguousfragment of at least 15 amino acids of SEQ ID NO:
 7. 3. The compound ofclaim 2, wherein the compound is PEGylated.
 4. The compound of claim 2,wherein the compound comprises a high affinity binding site for IgE. 5.The compound of claim 4, wherein the high affinity binding site isformed by the amino acid substitution D107E of SEQ ID NO:
 4. 6. Thecompound of claim 2, further comprising a pharmaceutically acceptablecarrier.
 7. A method for reducing a subject's immune response to anallergen, the method comprising: administering to a subject apharmaceutical composition containing an effective amount of a compoundselected from: a compound of formula X₁—R₀ (SEQ ID NO: 214), wherein R₀comprises SEQ ID NO: 4 and X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 7), or a contiguous fragment ofat least 15 amino acids of SEQ ID NO: 7, a compound of formula R₀—X₂(SEQ ID NO: 215), wherein R₀ comprises SEQ ID NO: 4 and X₂ comprisesSEGSAE (SEQ ID NO: 9), SEGSA (SEQ ID NO: 10), SEGS (SEQ ID NO: 11), SEG,SE, S, L, or —COOH, and a compound of formula X₁—R₀—X₂ (SEQ ID NO: 216),wherein R₀ comprises SEQ ID NO: 4, X₁ comprises at least 15 contiguousamino acids of TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQ ELEELRAEQQRLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO:7), and X₂ comprises SEGSAE (SEQ ID NO: 9), SEGSA (SEQ ID NO: 10), SEGS(SEQ ID NO: 11), SEG, SE, S, L, or —COOH; and a pharmaceuticallyacceptable carrier.
 8. The method of claim 7, wherein the pharmaceuticalcomposition is administered prophylactically to a subject at risk ofhaving an immune response to an allergen.
 9. The method of claim 7,wherein the pharmaceutical composition is administered to a subjectfollowing exposure to the allergen.
 10. The method of claim 7, furthercomprising administering steroid therapy.
 11. The method of claim 7,further comprising administering allergy shots to said subject.
 12. Themethod of claim 7, wherein the allergen is a food allergen, a pollen, aplant allergen, a dust mite, animal dander, insect stings, a fungus, aspore, a mold, latex, or a drug.
 13. The method of claim 7, furthercomprising a step of selecting a subject having an immune response to anallergen.
 14. A method for treating an IgE-mediated disease in asubject, the method comprising administering to a subject apharmaceutical composition containing an effective amount of a compoundselected from: a compound of formula X₁—R₀ (SEQ ID NO: 214), wherein R₀comprises SEQ ID NO: 4 and X₁ comprises TT QSLKQLEERA ARNVSQVSKNLESHHGDQMA QKSQSTQISQ ELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNERNEASDLLER LREEVTKLRM ELQVS (SEQ ID NO: 7), or a contiguous fragment ofat least 15 amino acids of SEQ ID NO: 7, a compound of formula R₀—X₂(SEQ ID NO: 215), wherein R₀ comprises SEQ ID NO: 4 and X₂ comprisesSEGSAE (SEQ ID NO: 9), SEGSA (SEQ ID NO: 10), SEGS (SEQ ID NO: 11), SEG,SE, S, L, or —COOH , and a compound of the formula X₁—R₀—X₂ (SEQ ID NO:216), wherein R₀ comprises SEQ ID NO: 4, X₁ comprises at least 15contiguous amino acids of TT QSLKQLEERA ARNVSQVSKN LESHHGDQMA QKSQSTQISQELEELRAEQQ RLKSQDLELS WNLNGLQADL SSFKSQELNE RNEASDLLER LREEVTKLRM ELQVS(SEQ ID NO: 7), and X₂ comprises SEGSAE (SEQ ID NO: 9), SEGSA (SEQ IDNO: 10), SEGS (SEQ ID NO: 11), SEG, SE, S, L, or —COOH; and apharmaceutically acceptable carrier.
 15. The method of claim 14, whereinthe IgE mediated disease is selected from the group consisting of:allergy, anaphylaxis, asthma, eczema, and rhinitis.
 16. The method ofclaim 7, wherein the subject's immune response to an allergen is achronic immune response to an allergen.
 17. The method of claim 7,wherein the subject is being treated with an allergy or anaphylaxistreatment.
 18. The compound of claim 2, wherein the compound does notcomprise amino acids 290-298 of SEQ ID NO:
 3. 19. The compound of claim2, wherein the compound does not comprise amino acids 290-321 of SEQ IDNO:
 3. 20. The method of claim 7 or 14, wherein any one of the compoundsdoes not comprise amino acids 290-298 of SEQ ID NO:
 3. 21. The method ofclaim 7 or 14, wherein any one of the compounds does not comprise aminoacids 290-321 of SEQ ID NO: 3.